The T lymphocyte carries a receptor specific to the antigen against which it is directed, called TCR. This TCR is composed of several chains, the α and β chains of which are involved in the specific recognition of a particular antigenic peptide presented in an HLA molecule. This recognition is shown by the ability of the α/β TCR of the T lymphocyte to bind with a certain affinity to HLA-peptide complexes present on the surface of the target cell. Reciprocally, soluble HLA-peptide complexes are capable of binding to the TCR present on the surface of T lymphocytes specific to the HLA-peptide complex in question.
At the present time, the system studied most at the molecular level is recognition by CD8+ T lymphocytes of antigenic peptides present in class I major histocompatibility complex (MHC) molecules, and in particular in the HLA-A0201 allele.
In this system, it has been established that the affinity of the TCR for the HLA-peptide complex is very low compared to the affinity of an antibody for its antigen. For this reason, detection of TCR-carrier lymphocytes which are reactive towards a specific peptide in this HLA context with the aid of soluble HLA-A0201 molecules which are charged with peptides and marked is impossible. To overcome this low affinity, Altman et al (1) prepared a multivalent reagent composed of HLA-A0201-peptide complexes where the heavy chain of the HLA is biotinylated, which allows combination as a tetramer with streptavidine. This HLA-peptide tetramer has an increased avidity for the appropriate TCR-carrier T lymphocytes and can therefore be used to visualize reactive populations by immunofluorescence.
However, the TCR is not the only molecule of the T lymphocyte which can interact with the HLA-peptide complex. In fact, during physiological recognition the binding of the TCR to the MHC-peptide complex is intensified by binding of the co-receptor CD8 to a constant portion of class I MHC molecules. The participation of CD8 in the interaction varies from one lymphocyte clone to the other and in some cases can lead to a very significant increase in the capacity for binding to a given HLA-peptide complex. This ability of CD8 to bind to class I HLA consequently leads to a background noise of binding of class I HLA tetramers on the CD8+ T lymphocytes which carry TCR which are non-specific to the HLA-peptide complex. This background noise increases with the concentration of tetramer used and can lead to falsely positive immunofluorescence results. To attempt to reduce this non-specific marking, the majority of teams carry out their marking with class I HLA tetramers in the presence of anti-CD8 antibodies. However, only some anti-CD8 antibodies are effective and the optimum concentration ratios between the antibody and the tetramer must be readjusted for each test. As a result of these disadvantages, detection of specific sub-populations with a low representation within a non-specific population (for example of the order of 0.1 to 1%) becomes difficult.
Another potential application of HLA tetramers has moreover been proposed. This comprises isolation by screening (in flow cytometry or by immunomagnetic screening) of lymphocyte populations which are reactive towards a given HLA-peptide complex for the purpose of in vitro expansion and then therapeutic use within passive anti-viral or anti-tumoral immunization protocols. However, the background noise of binding of the tetramer due to the participation of CD8 may constitute a serious obstacle in this application, since it leads to isolation of an often significant fraction of T lymphocytes which are not reactive with respect to the selecting HLA-peptide complex.
Salter et al (2) have shown that binding of a membrane HLA expressed by cells transfected with a CD8αα co-receptor was modified when the HLA carried a mutation in the α3 domain.
Study of such mutations by the inventors has led them to verify that soluble mutated tetramers effectively bind less CD8, regardless of whether αα or αβ, combined or not combined with a TCR on the surface of the T lymphocyte, which manifests itself in a reduction in the background noise.
It is therefore to be expected that the loss in affinity resulting from the mutation leads to a loss in the specific signal which is total or restricted to certain CD8-dependent T lymphocyte clones. In this respect, the article by Salter et al shows that certain CD8-dependent alloreactive clones lose their cytotoxicity with respect to cells carrying mutated HLA-A2, whereas others are less affected.
It would thus be possible that the mutated tetramers detect only a fraction of reactive cells (the less CD8-dependent) within a polyclonal population.
The numerous comparative markings of polyclonal populations with mutated and native tetramers carried out by the inventors by double-marking with an anti-CD8 antibody demonstrate that, on the contrary, the mutated tetramer unexpectedly recognizes the same percentage of specific cells as the native tetramer.
Furthermore, comparison of the marking with the mutated tetramer on a highly CD8-dependent clone and a clone of low CD8 dependency shows a comparable effectiveness of the binding of the tetramer with respect to the intensity of the expression of the TCR.
It thus seems that the mutation very significantly reduces binding of the tetramer to CD8 alone, but affects its binding to the TCR-CD8 complex much less.
The invention therefore lies in the utilization of the properties demonstrated in mutated or, more generally, modified HLA multimers and provides such multimers and their complexes with antigenic peptides, as new products.
It also provides the use of these molecules for the detection and/or isolation of peptide-specific CD8+ T lymphocyte populations.
It additionally provides a method for detection and/or isolation of such populations with the aid of such molecules which are charged with peptide, in particular for applications in diagnostics and therapeutics.
The multimers according to the invention are built up from recombinant protein analogues of class I MHC and are characterized in that the proteins comprise at least one modification in the zone of interaction of a heavy chain of the class I MHC with the CD8 co-receptor of T lymphocytes, leading to a reduction, or even suppression of the affinity of the interaction between the heavy chain and the CD8. The modification of the zone of interaction more specifically relates to the α3 domain of the heavy chain.
More particularly, it is a mutation in the α3 domain of at least one amino acid with respect to the corresponding domain of a native heavy chain which is capable of binding to the said CD8 co-receptor.
There may be mentioned by way of example the mutation of an alanine residue into a valine residue in position 245 of the α3 domain of the HLA-A2 molecule.